Fixation of tissue and cellular samples often makes protein-based epitopes inactive, or inaccessible for immunostaining due to protein cross-linking. Antigen retrieval (AR) is the process by which target epitopes are made accessible for immunostaining. Overcoming fixation-induced cross-linking allows for target epitopes buried within the tertiary structure of proteins to become accessible for binding with primary antibodies. Antigen retrieval also advantageously lowers the threshold for detection of antigens, thereby reducing the amount of antibody needed for detection, reducing background staining, and minimizing the occurrence of false negative results. Therefore, immunohistochemistry (IHC) and immunocytochemistry (ICC) protocols often include a pretreatment step to increase the intensity of immunostaining and to retrieve the antigen of interest.
Many existing pretreatment steps for immunostaining involve the incubation of the cellular or tissue sample of interest at high temperatures of about 80° C. or greater in various solutions (e.g., buffers, EDTA, acids, bases, surfactants) to prepare the sample for immunostaining. These types of sample pretreatment methods were largely developed and optimized for processing tissue samples. Tissues can withstand the high temperature of processing and maintain morphology because these tissues are often fixed in formalin and then embedded in paraffin prior to sectioning and IHC processing, and because the tissue sections maintain the support of the surrounding stromal tissue architecture. Cytology specimens are not fixed to the same degree as tissue samples, are not normally embedded in paraffin, and do not contain stromal support material to maintain cellular morphology during high heat pretreatment. Thus, high heat pretreatment methods that serve to increase epitope exposure and accessibility to the primary antibody degrade the cellular morphology of cytology specimens.
Therefore, pretreatment methods and compositions that are effective in antigen retrieval, yet maintain cellular morphology, are needed for the processing of cytology samples, in particular, in preparation for immunostaining.